Method of separating chlorophyll from vitamins and other fat soluble materials



0f l, 1946- w. R. GRAHAM, JR., E-rm. 2,408,625

METHOD 0F SEPARATING CHLOROPHYLL FROM VITAMINS AND OTHER FAT SOLUBLE MATERIALS Filed June s, 1942 chlorophyll in the cake.

Patented Oct. 1, 1946y METHOD F SEPARATING CHLOROPHYLL FROM VITAMINS AND. OTHER FAT SOLU- BLE MATERIALS William R. Graham, Jr.,

0. Kohler, Mission, Ka Hoover, Kansas City, Mo., can Dairies Incorporated,

Kansas City, and George ns., and Richard D.

assigner: to Ameri- Kansas City, Mo., a

corporation of Maryland, and The Quaker Oats Company, Chicago, lll., a

Jersey corporation of New Application Jane 5, 1942, sensi No. 445,958

(el. 21o-8.5)

4 Claims.

1 Our invention relates paratus for separating and concentratingcarotene, vitamins, xanthophyll, and other fat soluble organic material from chlorophyll and other nondialyzable solutes in plants such as grass, al-

falfa, spinach, or other vegetable matter. The process may alsol be employed to separate and concentrate the carotene, vitamins. xanthophyll and other iat soluble organic material from other non-dialyzable materials in plants containing n'o chlorophyll such as carrots or sweet potatoes.

We have found that certain fractions of a fat soluble extract from plants will dialyze through a rubber or synthetic rubberv membrane while other fat soluble fractions in the solution will not. For instance, if all of the fat soluble materials are extracted from a suitable plant material. the carotene, vitamins and certain other materials will pass through the membrane while the chlorophyll and certain other materials will not diifuse through the membrane. When extracting carotene, vitamin K, xanthophyll, and-other fat soluble material from the plant material, it is desirable to remove the chlorophyll for the reason that chlorophyll. while harmless. imparts an un- ,desirable green color 'to the vitamin solution. Also, when isolated in its pure form, chlorophyll is a valuable byproduct.

Sterols both from plant and animal sources will also pass through the rubberv or Synthetic rubber membrane. Thus. this method may also be used to separate sterols from other iat soluble materials.

Many of the materials in the chloroplastV are labile and are destroyed or altered by addition oi certain chemicals. This characteristic of these materials has made their separation and isolation a difficult task.

' In the past, theSchertz process has been extensively used. A description of this process may be found in volume 30, page 1073 of Industrial 8: Engineering Chemistry, published in 1930. The Schertz process employs a hydrocarbon solvent to extract the carotene leaving the bulk of the Then an 85% acetone extraction is employed to remove the chlorophyll. One disadvantage of this method is that it requires two separate extractions of the meal with different'solvents. ,Thus two separate solvent vreto a method ofand alJ- l ral moisture content from the A tion will be apparent covery systems must be used and the total quantity-oi solvents is disproportionatelylarge. Another difiiculty with this process is that the rst extraction is notsulciently selective" and considerable amounts oi chlorophyll are extracted with the carotene.

We have discovered a method of separating chlorophyll from vitamins which requires the use of only one solvent. A suitable plant mate` rial, such as grass, alfalfa or spinach, which contains vitamins and chlorophyll, is treated in this manner. The plant material is iirst partially dried to remove a substantial part of the natuplant cells. The dried material is pulverized and then extracted with a fat solvent such aspentana'hexane, heptane, petroleum ether, benzene, acetone, butyl alcohol, ethylene dichloride, or other fat solvents or mixtures of such solvents. This solution, preferably in relatively dialyzed through a membrane madekof rubber, synthetic rubber, or the like, into a fresh solvent. The carotene, vitamins, lxanthophyll and other iat soluble materials pass through the membrane into the fresh lsolvent while the bulk oi the chlorophyll remains behind. An important object of our invention, therefore, is the provision of a novel method of separating carotene, vitamins and other fat soluble materials from chlorophyll and other materials that requires the use of but one solvent.

Another object of our invention is the provision of a method of the above mentioned character that may be easily yand inexpensively practiced.

Other objects and advantages of vour invenduring the course oi the following description.

In the drawing forming a part of this specification and wherein like numerals are employed to designate like parts throughout the same:

Fig. 1 is a side elevation of an apparatus that may be used to practice our process, parts of the apparatus being shown in section for clearness of illustration, and

Fig. 2 isa side elevation of another form vof. apparatus that may be used to practice our process. parts being shown in section and parts in elevation` concentrated form, is then near the bottom 'the separatory funnel I1- and is Referring'now to the apparatus shown in Fig. 1, Athe numeral I designates a separating tank adapted to be filled with a fat solvent such as heptane. Flask II also contains a quantity of the selected solvent. The contents of the flask are heated by an electric element I2. When the solvent in flask II is heated, the vapors rise through tube I3 and into the upper portion of tube I4 which extends through a condenser I5. As the vapors enter tube I4 they condense and gravitate downwardly the tube which extends into and has its lower end of separating tank I8. Thus, flask II will continuously supply tank III with fresh solvent. An overflow tube I6, having a through the lower portion of trap portion I6a, connects the upper end of tank I0 with tube I3. The overflow tube is preferably tilted slightly from the horizontal so that overflow liquid from tank I8 will pass through tube I6 into pipe I3 and back into ilask II. A concentrated solution of plant material is introduced at conducted through connecting pipe I8 to a rubber or syntheticrubber tube coil I9. Neoprene tubes are conventional and satisfactory.

The lower or discharge end of the coiled tube I9 is connected to tube 20 which discharges ,into a flask 2|. Container III and ask I I are equipped with suitable Stoppers assuring liquid tight connections around th'e inlet and discharge tubes. Introduction of the plant concentrate to the systern is controlled by valve 22.

As a typical example of the method as practiced in the apparatus shown in Fig. 1, a quantity of alfalfa was passed through a drum type dryer having an intake temperature of approxl. mately 1600 F. and an outlet temperature of approximately 250 F. 'I'he green plant material remained in the dryer only sufllciently long to remove a substantial amount of the natural plant moisture without material discoloration or charring of the chlorophyll. This preliminary drying step is not essential but we have found that it facilitates the later separation of the chlorophyll fraction from the other fat soluble materials.

'I'he dried plant material was then passed through a hammer mill and ground to a flne powder. After grinding, th'e powdered plant material or tane to dissolve the fat soluble materials from the plants. This solution was then passed through a still to remove part of the solvent leaving the solution in relatively concentrated form. 250 ml. of this concentrated solution was then poured into separatory funnel I1. Valve 22 was opened and the concentrate permitted to flow slowly through tube I8 and coiled tube I9. Slmultaneously fres 'solvent was introduced to the bottom of the tank I8 through pipe I4 and was `circulated around the exterior of the coiled tube I9 overflowing from the tank through pipe I6 back to flask II. Throughout the entire operation, heater I2 causes a constant circulation of solvent through the apparatus as previously explained. As the plant extract passes slowly through neoprene tube I9, the carotene, vitamin K, xanthophyll, etc., diffuse through the wall of the neoprene tube and into the heptane solvent in tank I8. The dilfusate containing the vitamin material overflows tank I 0 through pipe I4 into flask II.

'I'he chlorophyll and other non-dialyzable materials are retained within neoprene tube I9 and are discharged as a dialyzate through tube pigment was mixed with sufficient hep- About ve hours is required of solution through the flask with fresh solvent. The dialyzate in flask 2 I, which is a dark green color and contains practically all of the chlorophyll, is then poured back into separatory funnel I1. Satisfactory results are obtalnedif th'e plant material is passed approximately six times through the apparatus.

The following table shows the results of an actual experiment conducted as outlined above.

Carotene Chlorophyll laltio, Sample Total in T mlm ch tiro' Percent o Percent p yl to fraton, recovery imgen, recovery carotene 1st distinta-. 70.11 23. 4 13s 2. c 1. 9c 2nd dinusate 64. s 21. 5 167 a. 1 2. 5s 3rd difrusate- 47.0 15. s 116 2. 2 2. 47 4th ditrusata 5s. a 19. 3 172 a. 2 2. 95 5th difrusate. 33. s i1. 2 154 2. 9 s. os 6th diffusera.. 13.8' 4.6 10o 1.9 7.25 Total (Iilusates 288.3 9st 847 15.9 2.94 Dialyzate. 13. 2 4. 4 4, 536 s4. 1 343. oo

It should be noted that 95.6 per cent of the carotene (pro-vitamin A) in the solution was recovered, while` only 15.9 per cent'of the chlorophyll diifusate passed through tube 19. A biologicaichick assay indicated that 98.5 per cent of the total vitamin K present was recovered in the dlflusate. Qualitative analyses have demonstrated that a high percentage of the xanthophyll also diffuses through tube I9.

Explaining now the operation of the apparatus shown in Fig. 2, wherein a series of membranous sheets or films are used in place of the neoprene tube I9 in Fig. l. This apparatus operates more rapidly and handles greater quantitiesl of plant material than the apparatus shown in Fig. 1. Describing now the portion of the apparatus that permits a constant circulation of liquid solvent. An essentially rectangular tank 23 is dlvided into chambers, 25,4 26, 21, and 28 by membranes 29 of rubber or synthetic rubber. A flask 30 is heated by an electrical heating element A tube 32 extends from flask 30 and connects with a pipe 33. The upper portion of pipe 33 is enclosed by a condenser 34 and the lower portion of the pipe extends into chamber 24. I

Siphon tube 35 connects chamber 24 with chamber 26 and siphon tube 36 connects chamber 26 with chamber 28. A siphon tube 31 connects chamber 28 with a constant level device 38. Liquid discharged into the constant level 'device flows through trap 39a in tube 39 to flask 38. Ch'ambers 24, 25, 26, 21 and 28, constant level devices 38 and 45, and flask 38 are filled with a solvent such as heptane. When the apparatus is set up, the siphon tubes 35, 36, 31, 43 and 44 are also lled with the solvent so that a siphoning action can take place. 'I'he liquid in flask 30 is heated. Vapors passing olf through pipe 32 are condensed and the condensate discharged into chamber 24. As the liquid in chamber 24 tends to rise, hydrostatic pressure forces liquidffrom the bottom of the chamber to pass through siphon tube'35 to chamber 26, from chamber 26 the liquid passes through siphon tube 36 to cham-V amaca The crude plant extract or concentrate,

duced through separatory funnel 45, is allowed' to lilow through tube 4I controlled by valve 42, into chamber 21. A siphon tube 4I connects chamber 21 with' -chamber 25. A siphon tube 44 connectsV chamber 25 with a constant level device 45. Liquid delivered to the constant level device 45 overiiows into tube. 45 which conducts it into a flask 41.

As an example of a typical operation, a concentrated heptane solution of plant material prepared in the manner hereinbefore described was placed in container 40. In actual practice, container 40 is much larger than shown in the drawing. 'I'he heptane solvent was then placed in the apparatus as described above. A constant circulation of the solvent was effected by heating element 3|. Valve 42 was opened suiliciently to permit the extract in container 40 to slowly enter chamber 21 through tube 4|. As the solution of plant material tends to rise in chamber 21, the carotene, vitamin K, xanthophyll, etc., diffuse through rubber membranes 29 into chambers 26 and 28 while the chlorophyll and other non-dialyzable materials will remain in chamber 21 and be transferred to chamber 25 through siphon tube 43. vThe vitamins entering the solvent in chamber 28 will pass through tubes I1 and 39 into ask 35. Vitamin materials diffused into chamber 25 pass into chamber 28 through siphon tube l35. Vitamins remaining in chamber 21 pass into chamber 25 through tube 43 where dialysis again takes place. In chamber 25, the carotene, xanthophyll and vitamins again diiluse through membranes 29 and are taken up by the solvent in chambers 24and 25. The materials not diffused through the membranes pass off from chamber 25 through pipe 44 into constant level device 45, thence into flask 41 through pipe 45. Thus, the carotene, xanthophyll and vitamins accumulate in flask 30, while the chlorophyll and other non-dialyzable materials accumulate in ilask 41. Solvent in the vitamin and chlorophyll solutions is then removed by processes well known to the art as by passing live steam through the residue under Ivacuum after distillation. The greater membrane surface of this apparatus greatly decreases the time required to separate the crude chlorophyll from the other fat soluble materials. This apparatus also permits the use of thinner membranes than is possible where a coiled tube is used to speed up dialysis. We have kfound that three passes of the dialyzate are generally sumcen't to obtain a satisfactory separation. To recirculate the plant material, the dialyzate is removed from flask 41 and returned to separatory funnel 40. The results of a n in the following table.

this apparatus are given Carotene Chlorophyll clilitio, sample Total in n gitt- Peroent Total Percent p y o fmffon' recovery fraction recovery carotene ist diflusate.-- 48. 7 583 5. 8 1. 9 2nd diusate-- 124 21. 6 361 3. 9 2. 9 3rd diusate- 102 17. 7 464 5. 0 4. 6 Total diflusrites 506. 0 88. 0 1, 358 14. 7 2. 68 Dialyzate. 68. 6 l2. 0 7, 870 3 114. 6

It will be noted that 88 per cent of the carotene was recovered in three passes of the plant solution through the apparatus and that only 14.7 per typical experiment using capable of diffusing cento! the chlorophyll was recovered with the carotene.

We have tried man3 types of dialysis membranes but have found rubber or synthetic rubber membranes to be most satisfactory. We have found thata vulcanized rubber membrane stands up against the solvent longer lthan a natural rubber such as latex. The following membranes were tested and, in each instance, it was found that very little,'if any, of the carotene or xanthophyll dialyzed through the membrane; collodion, paper parchment, animal parchment, pliofllm, lucite, cellulose acetate, animal intestine, animal intestine (bile salts added) fiber tubing, paper impregnated with gelatin, and Cellophane.

A number of iat solvents have been found to be satisfactory. Among the solvents tested were -petroleum ether, acetone, ethylene dichloride, benzene, butyl alcohol, and various of the normally liquid homologs of hexane, including pentane, hexane, and heptane.

It may thus be seen that we have accomplished the objects of our invention. We have provided a highly eilicient method of separating carotene, vitamins, xan-thophyll, etc. from a fat solvent extract of plant materials. We have provided suitable apparatus for practicing the process. vThe process requires the use of but one solvent and by using theone solvent a high percentage of vitamin yield is obtained. The process may be easily and inexpensively practiced.

It is to be understood that the forms of the invention herewith shown and described are to be taken as preferred examples of the same and that changes may be made in the apparatus and method which do not depart from the spirit of our invention or the scope of the appended claims. Y

Having thus described our invention, we claim' 1. A method of separating fat soluble vitami. and pro-vitamins from extraneous organic ma terial contained in a fat solvent extract of plant materials by dialyzation utilizing a membrane the vitamins and pro-vitamins, comprising the steps of passing the fat solvent mixture and a relatively uncontaminated fat solvent on opposite sides of the membrane to selectively diffuse the vitamins and pro-vitamins through the membrane into the uncontaminated solvent, separately removing the vitamin and provitamin containing solvent and the solvent containing the organic materials, and recovering the vitamins and pro-vitamins from its solvent.

2. A method of separating fat soluble vitamins and pro-vitamins from chlorophyll contained therewith in a fat solvent extract of plant materials by dialyzation utilizing a membrane capable of diffusing the vitamins and pro-vitamins, comprising the steps of passing the fat solvent mixture and a relatively uncontaminated fat solvent on opposite sides of the membrane to selectively diffuse the vitamins and pro-vitamins through the membrane into the uncontaminated solvent, separately removing the vitamin and pro-vitamin containing solvent and the solvent containing chlorophyll and recovering the vitamins and provitamins from its solvent. .l

3. A method of separating fat soluble vitamins and pro-vitamins from extraneous organic material" contained therewith in a fat solvent extract of plant materials by dialyzation utilizing a membrane selected from the class consisting of rubber and synthetic rubber, comprising the steps of passing the fat solvent mixture and a relatively uncontaminated fat solvent on opposite sides of .the membrane to selectively diiuse the vitamins and pro-vitamins .through the membrane into the uncontaminated solvent, separately -removing the vitamin and pro-vitamin containing solvent and the solvent containing the organicl materials and recovering the vitamins and provitamins from its solvent.

4. A method of separating fat soluble vitamins and pro-vitamins from chlorophyll contained therewith in a. fat solvent extract of plant ma.- terials by dialyzation utilizing a, membrane selected from the class consisting of rubber and syn- 

